REVIEW A broadening view of recombinational DNA repair in

Michael M. Cox* Department of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA

Recombinational DNA repair is both the most complex and least understood of DNA repair pathways. In bacterial cells grown under normal laboratory conditions (without a DNA damaging treatment other than an aerobic environment), a substantial number (10–50%) of the replication forks originating at oriC encounter a DNA lesion or strand break. When this occurs, repair is mediated by an elaborate set of recombinational DNA repair pathways which encompass most of the involved in DNA metabolism. Four steps are discussed: (i) The replication fork stalls and/or collapses. (ii) Recombination enzymes are recruited to the location of the lesion, and function with nearly perfect efficiency and fidelity. (iii) Additional enzymatic systems, including the fX174-type primosome (or repair primosome), then function in the origin- independent reassembly of the replication fork. (iv) Frequent recombination associated with recombinational DNA repair leads to the formation of dimeric chromosomes, which are monomerized by the XerCD site-specific recombination system.

contribution to bacterial DNA replication under Introduction normal growth conditions. A summary of the likely Recombinational DNA repair represents a cross-roads fate of a replication fork in the where virtually every aspect of DNA metabolism comes chromosome can serve as an overview to organize this together. When a bacterial cell is subjected to UV discussion (Fig. 1). Once initiated at oriC, some irradiation or other DNA damaging treatment, DNA replication forks complete their task, while others replication rapidly comes to a halt. After 30–40 min, encounter either an unrepaired DNA lesion or a DNA replication is restored to its original level. Replication strand break at a lesion undergoing repair. At these restart (Khidhir et al. 1985; Echols & Goodman 1990; encounters, the replication complex halts and/or Echols & Goodman 1991) requires both recombination collapses. The resulting gap or double-strand break is and replication functions. In the meantime, a wide array processed by recombination enzymes. The branched of DNA repair processes are induced as part of the SOS DNA replication fork is re-established after a lag of system, including many that facilitate the recombina- some minutes. A replication complex which may be tional DNA repair and replication restart. Information distinctly different from that assembled at oriC, comes about what occurs during the 30–40 min required for together in an origin-independent manner, and replica- replication to recover is still limited, but available tion again proceeds unimpeded. Any DNA lesions left experimental data can provide some insight. behind are now within double-stranded DNA and can The transient abatement of DNA synthesis is almost be processed by excision repair pathways. The improper certainly not limited to environmental stress producing resolution of recombination intermediates (Holliday unusual levels of DNA damage. Instead it is a structures) producing a dimeric bacterial chromosome manifestation (albeit dramatic and readily observed) of is countered by a distinct and specialized site-specific a process that makes an important and regular recombination system. The pathways presented in Fig. 1 are not intended * Correspondence: E-mail: [email protected] to be comprehensive or unique, and the precise

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primarily to address the requirements of recombina- tional DNA repair. The importance of this process is also oriC - dependent replication reflected in an extraordinary concentration of facilitating (DNA polymerase III DnaB, DnaC, DnaG) sequences (chi sites) seen throughout the E. coli . A concise integration requires a focus on several GAP repair DS break repair themes. First, a premature termination of replication fork movement which requires recombinational DNA DNA lesion DNA nick repair is a very common occurrence even in the absence of 1 Replication fork 1 Replication fork disassembly collapse treatments designed to elevate DNA damage. Second, the re-initiation of replication after recombinational repair requires a specialized enzymatic system and mechanisms distinct from those applied at the genomic origin. Recombination Recombination Third, the organization of the replication fork may be 2 (RecA + RecFOR) 2 (RecBCD RecA) altered after recombinational DNA repair. Fourth, homologous is a required step in this repair pathway, bringing with it an array of genomic and cellular consequences. resolution resolution (RuvABC + RecG) (RuvABC or RecG) The paradigm outlined in Fig. 1 can enhance our 3 understanding of the and biochemistry of the Origin-independent replication proteins playing a direct or indirect role in recombina- re-start tional DNA repair. It also has the potential to illuminate Repair Primosome a surprising number of biological systems, enzymatic activities and cellular phenomena that have sometimes been difficult to place in an appropriate functional origin-independent replication 4 -Termination context. -Dimer resolution (XerCD) Figure 1 Pathways of recombinational DNA repair. The steps described in the text are outlined. The listing of proteins involved The structure of replication forks assembled at in each step is not meant to be exhaustive. Circled numbers oriC correspond to the four steps highlighted in the text. Following an oriC-dependent initiation process that relies on the activities of the DnaA and DnaC proteins, replication forks proceed bidirectionally from the E. coli mechanisms by which these processes occur are left origin (Kornberg & Baker 1992; Marians 1992). The intentionally vague. Many of the ideas in Fig. 1 can be contiguous protein complex at each fork consists of the traced to recombinational repair models presented by asymmetric DNA polymerase III holoenzyme and the West and Howard-Flanders (West et al. 1981) and Szostak DnaB (Fig. 2). The DnaG plays an et al.(Szostaket al.1983).Manyofthesameideashavealso intermittent role in the priming of lagging strand DNA been developed in a number of recent articles, books and synthesis (Tougu & Marians 1996). Auxiliary proteins reviews (Zavitz & Marians 1991; Cox 1993; Livneh et al. (DNA topoisomerases, single-strand DNA binding 1993; Asai et al. 1994a; Friedberg et al. 1995; Sherratt et al. protein (SSB)) play important roles that do not 1995; Kuzminov 1996a; Kogoma 1997; Roca & Cox necessarily require direct physical contact with the 1997). Drawing from different perspectives, each con- replication fork complex. The result is an integrated tributes to the synthesis attempted here and should be complex that carries out DNA synthesis on both DNA consulted for more detailed discussions of individual template strands (Fig. 2). points and for some alternative views. Continuous DNA synthesis on the leading strand is Bacteria have made an extraordinary evolutionary complemented by the coordinated synthesis of Okazaki investment in recombinational DNA repair. At some fragments on the lagging strand. The DnaG primase point in the scheme of Fig. 1, almost every bacterial interacts transiently with the DnaB helicase (Tougu & protein known to play a role in DNA metabolism leaves Marians 1996) to effect routine priming of lagging its mark. Many of these proteins, particularly those with strand DNA synthesis. For replication which is initiated the designations Rec, Pri and Xer, may have evolved at oriC, the DnaB and DnaG proteins constitute a

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A 5' minimal (oriC-type) lagging strand primosome, both in 3' vivo and in vitro (Kornberg & Baker 1992; Marians 1992). Both of these proteins are essential in E. coli, and 3' ts mutants exhibit a rapid-stop phenotype with respect core 5' γ τ leading strand to DNA synthesis (Wechsler & Gross 1971). The DnaB complex protein is the only one of the multiple E. coli DnaB core lagging strand that is absolutely required for chromosomal replication helicase (Baker et al. 1986). RNA primer Conspicuously absent from the complex at the RNA primer replication fork are five of the proteins defined as 5'3' components of a larger E. coli primosome, often primase referred to as the fX174-type primosome. These B include the PriA, PriB, PriC, DnaC and DnaT proteins, originally discovered during in vitro studies of the replication of the fX174 genome (Arai DnaB helicase & Kornberg 1981; Zavitz & Marians 1991). This more elaborate primosome is required for initiation of

DnaG replication and lagging strand synthesis in bacteriophage primase fX174 and also for plasmids with ColE1 origins (Zavitz new & Marians 1991). However, a fX174-type primosome RNA primer is not required for replication originating at oriC, either in vivo or in vitro. This opens the intriguing question of the cellular function of a fX174-type primosome, which presumably did not evolve to serve the needs of β primase or plasmids. C The PriA protein plays a central role in the assembly of the fX174-type primosome, and also exhibits DNA- dependent ATPase and DNA helicase activities in vitro (Wickner & Hurwitz 1975; Shlomai & Kornberg 1980). The issue of primosome function has been addressed in the isolation of mutant cells deficient in New β subunit PriA activity (Lee & Kornberg 1991; Nurse et al. 1991). loaded at RNA primer Cells lacking PriA exhibit growth defects, filamenta- tion, and an induction of the SOS response. They have a reduced viability, but the mutation is not lethal (Nurse et al. 1991). Combining the priA defect with an sulA mutation that relieves the filamentation phenotype, resulted in cells with improved viability and growth rates that were 60% of wild-type (Nurse et al. 1991). A D priA mutation that eliminated the ATPase function but retained the primosome assembly activity restored cell

Figure 2 A replication fork assembled at oriC. (A) An

New β subunit asymmetric DNA polymerase III complexed with the DnaB helicase moves along the DNA. (B) At intervals, the DnaG primase binds to the DnaB helicase and synthesizes an RNA primer on the lagging strand. (C) The g complex them loads a new b subunit complex at the new primer. (D) Once the synthesis of one Okazaki fragment is complete, the old b subunit complex is released along with the replicated DNA, and a new Discarded β subunit Okazaki fragment is initiated at the new RNA primer utilizing the new b subunit complex.

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growth and viability to normal levels (Zavitz & Marians example, the introduction of a damaged but unrepli- 1992). Overall, the present work indicates that oriC- cated viral or plasmid genome into a cell does not dependent replication does not depend on a functional induce SOS (D’Ari & Huisman 1982; Sommer et al. PriA protein in vivo. The reconstitution of oriC- 1991). The general conclusion of many studies is that dependent DNA replication has demonstrated no replication forks stall at the sites of DNA damage and need for PriA protein in vitro as well (Baker et al. that single-strand gaps open up in the DNA as a result. 1986; Funnell et al. 1987). The gaps provide binding sites for RecA protein, which It has been recognized for some time that replication in turn leads to an induction of the SOS response. forks might be halted at DNA damage (Kuzminov Alternatively, and at least as important, the replication 1995a). Complementing many other experimental forks might encounter DNA strand breaks and thereby paths, indicating a link between recombination and generate double-strand breaks. As outlined below, DNA repair (Cox 1993; Clark & Sandler 1994; double strand breaks may form at stalled replication Kuzminov 1996a; Roca & Cox 1997), the analysis of forks. The processing of the DNA ends for recombina- priA mutants led to the suggestion that replication forks tional repair could also lead to RecA protein binding originating at oriC might be halted at DNA damage and SOS induction, as well as to DNA repair (Anderson and disassemble surprisingly often (Zavitz & Marians & Kowalczykowski 1997a,b). The same RecA protein 1992). The fX174-type primosome would then that binds to single-stranded DNA and induces the SOS function to re-establish a viable replication fork to response also plays a direct role in repair. It is important complete the DNA synthesis. In effect, the replication to note that it is the recombinational activities of RecA fork that completed DNA synthesis would often be that are required for recombinational DNA repair, as distinct from the replication fork generated at oriC. opposed to the RecA-mediated induction of repair Although this is not the only insight leading to a broader functions associated with the SOS system (Smith & synthesis of recombinational DNA repair pathways, it Wang 1989; Roca & Cox 1990; Asai et al. 1993). serves as an entry point for a more detailed examination of the steps outlined in Fig. 1. Quantifying step 1 under normal growth conditions What happens if the cells are not subjected to a DNA Step 1: the replication fork is halted at DNA damaging treatment? Under normal aerobic growth conditions, an E. coli cell suffers 3000–5000 DNA damage lesions of oxidative origin per cell per generation (Park The first suggestion that a replication fork might et al. 1992). Good estimates are not available for other collapse at the site of a DNA strand break came in types of damage, although oxidative damage is almost 1974 (Skalka 1974). The general idea that replication certainly the major source of lesions in the absence of fork progress is halted by many types of DNA damage is other environmental challenges. The effects of oxidative now supported by an array of experimental observations damage are evident in the phenotypes of many bacterial (Kuzminov 1995a). The response to a UV challenge strains lacking key activities in DNA metabolism. For provides ample evidence that damage halts the progres- example, recB polA or recA xth double mutants are sion of replication forks. As already noted, UV triggers a nonviable when grown aerobically, but survive under transient pause in DNA synthesis (Livneh et al. 1993). anaerobic conditions (Morimyo 1982; Imlay & Linn DNA fragments produced after UV irradiation have 1986). sizes that correspond to the average inter-dimer distance Although most lesions are repaired quickly, a in template strands, as though the replication forks substantial number of replication forks have their halted, then started up again so as to leave disconti- progress arrested at DNA damage. Without DNA nuities (Sedgwick 1975; Youngs & Smith 1976). The damaging treatments, and under conditions which are resulting single-strand gaps almost certainly represent generally used for the growth of bacteria in the the signal for induction of the SOS response (Livneh laboratory, it can be estimated that 10–50% of the et al. 1993; Friedberg et al. 1995; Kuzminov 1996a), and replication initiations at oriC are not completed studies of the requirements for SOS induction can without such interruption. This estimate is derived therefore reinforce the link between the appearance of from a variety of indirect observations. Some of the best the gaps and replication. A variety of reports have clues can be derived from studies of cells lacking the demonstrated that replication is required for the recA or recBC functions. induction of SOS (Sassanfar & Roberts 1990; Friedberg If a replication fork encounters a lesion at a stage in et al. 1995). DNA damage alone is insufficient. For repair in which a DNA strand break exists, the

68 Genes to Cells (1998) 3, 65–78 ᭧ Blackwell Science Limited Recombinational DNA repair in bacteria encounter will result in the formation of a double strand underline that these events are common and highly break that cannot readily be repaired in a recA or recBC significant to bacterial DNA metabolism. To the extent mutant. A careful look at linearized E. coli chromosomal that alternative pathways for the repair of stalled DNA (Michel et al. 1997) indicated that double strand replication forks exist that are independent of RecA breaks appeared in about 15% (after correction for and/or RecBCD (Cao & Kogoma 1995; Bi & Liu background) of the chromosomes of a recB or recC 1996; Saveson & Lovett 1997), the frequencies mutant, and over 20% of the chromosomes from a recA estimated from the DNA structures seen in recA and recD double mutant. In this study, cells were grown recBC cells could underestimate the severity of the aerobically in a minimal media. The frequency of problem. In addition, the frequencies are likely to vary double strand breaks can be interpreted as one measure greatly as a function of growth conditions. The presence of the encounters between replication forks and or absence of oxygen is a major factor. Even the choice genomic strand breaks, or other replication-associated of media, to the extent that it affects oxidative events that generated double strand breaks. metabolism (Galitski 1996; Roca & Cox 1997), may Additional studies of RecA-deficient cells yield alter the fate of replication forks. Finally, the similar clues. A defect in DNA replication and cell most straightforward message of the cell survival division in recA cells was first reported by Inouye curves published in numerous studies of rec mutants (Inouye 1971). Over 50% of the cells in a culture of a should be noted: even a modest challenge with a recA null mutant are nonviable under at least some DNA damaging agent kills the vast majority of rec¹ conditions (Capaldo et al. 1974). The frequency of cells (Livneh et al. 1993; Clark & Sandler 1994; productive replication initiation at oriC is significantly Kowalczykowski et al. 1994; Friedberg et al. 1995; reduced (Skarstad & Boye 1988). More than 10% of recA Kuzminov 1996a; Roca & Cox 1997). More systematic cells contain no DNA (Zyskind et al. 1992; Horiuchi & studies of the status of the bacterial chromosome as a Fujimura 1995), and many more have abnormal function of growth conditions and the presence or numbers of chromosomes (Skarstad & Boye 1988; absence of rec mutants could be highly informative. A Skarstad & Boye 1993). The observation of anucleate fascinating corroboration of these estimates for the cells has been explained as reflecting a defect in frequency of replication fork collapse and recombina- chromosomal partitioning (Zyskind et al. 1992), but tional DNA repair is evident in recent work on the chromosome loss due to the degradation of chromo- XerCD site-specific recombination system (step 4, somes with breaks provides a quantitative explanation below). for the appearance of anucleate cells as well as those with odd numbers of chromosomes (Skarstad & Boye Step 2: replication gives way to recombination 1993). These studies were carried out with bacterial cells grown aerobically in a variety of standard media. The pathways of Fig. 1 are an oversimplification in A stalled replication fork can lead to a double strand more than one respect. There are doubtless more than break (Kuzminov 1995b). Artificially halting replication two pathways for recombinational DNA repair, along forks (by including a ts mutation in a replication helicase with overlapping pathways and pathway variants. There like DnaB) increased the amount of linearized DNA are also many more steps and proteins involved in observed in recBC mutant cells (Michel et al. 1997). The individual pathways. The recombination functions of increase was not observed without replication. Placement recombinational DNA repair can be viewed as an of a replication termination site (ter) at the lac operon so adaptable and changing assemblage that can address a as to halt replication prematurely leads to a substantial wide range of DNA structural realities. The hierarchy of increase in the generation of anucleate cells in recA strains pathways and enzymatic activities defined to date for (Horiuchi & Fujimura 1995). This is consistent with the conjugational and transductional recombination (Smith generation of double-strand breaks at the stalled replica- 1991; Clark & Sandler 1994; Kowalczykowski et al. tion forks followed by a degradation of the broken 1994) reflect the DNA substrates presented to the cell chromosomes. High levels of homologous recombina- under those specialized conditions, and need not be tion have been noted in the region near the terminus of exactly replicated in recombinational DNA repair. For replication (Louarn et al. 1994), an observation that can example, the DNA damage sensitivity conferred by be explained by the introduction of breaks at the site of mutations in RecF pathway mutant genes (Roca & Cox stalled replication forks (Kuzminov 1995b). 1997) suggests that this pathway may be more The estimate of 10–50% for the premature arrest of important to recombinational DNA repair than it is to replication forks is obviously approximate, but serves to conjugational recombination in wild-type cells. New

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assays examining recombination between chromosomal to prevent extensive degradation of the chromosome by direct repeats also suggest that sexual recombination RecBCD. The chi sites also appear to be located within assays under-estimate the importance of the RecF islands of sequences shown to be preferred DNA binding pathway (Galitsky & Roth 1997). sites for the RecA protein (Tracy et al. 1997). The Once a replication fork halts at a DNA lesion or evolution and conservation of this highly facilitative encounters a DNA strand break, it is believed to positioning of chi sites in the E. coli genome can be viewed disassemble (Kuzminov 1996a). Direct evidence for this as indirect evidence that most double-strand breaks, outcome is limited. DNA polymerases are halted by a subject to recombinational DNA repair in bacteria, are variety of DNA lesions in vitro (Livneh 1986; Banerjee generated in the course of replication. et al. 1988; Lawrence et al. 1990; Bonner et al. 1992). The repair of DNA gaps which are generated when a The interruption is followed by polymerase dissociation replication fork encounters a DNA lesion follows a (Livneh 1986). However, these are simple model pathway dependent on the RecF, RecO and RecR systems that do not reproduce a complete replication proteins (Fig. 1). At least one major function of these fork. At a minimum, the disassembly of a replication proteins is to modulate the assembly of RecA protein fork upon encountering a nick or lesion is logical. The filaments in the single-strand gap. RecA filaments available evidence is insufficient to preclude the assemble and disassemble 50 to 30 in an end-dependent possibility that stalled replication forks remain partially fashion, with a protein being added at one end and or entirely intact under some circumstances. deleted at the other (Roca & Cox 1997). Certain Repair of the double-strand break resulting from an mutants of RecA protein suppress the defects of encounter with a nick is dependent on the RecBCD recFOR mutants (Thoms & Wackernagel 1988; and follows a pathway outlined in Fig. 1. Large Madiraju et al. 1992; Wang et al. 1993). In vitro work parts of this proposed pathway have been reconstituted to date indicates that the RecR protein forms in vitro (Kowalczykowski 1994; Anderson & Kowalczy- alternative complexes with the other two proteins, kowski 1997b; Eggleston et al. 1997). In short, the with different functions (Fig. 3). The RecOR complex RecBCD enzyme binds to a double-stranded DNA facilitates the binding of RecA protein to SSB-coated end, unwinds the DNA and degrades the two strands DNA (Umezu & Kolodner 1994), and prevents the asymmetrically, with the 50-ending strand remaining end-dependent disassembly of the RecA filament (Shan relatively intact. Upon encountering the 8- et al. 1997). The RecFR complex binds primarily to sequence called chi, the enzyme’s 30 to 50 double-stranded DNA and can prevent excessive activity is abated, a 50 to 30 exonuclease is up-regulated, extension of the filament into the adjoining duplex and RecBCD facilitates the loading of RecA protein on DNA (Webb et al. 1997). If this activity of RecFR to the prepared single strand (Anderson & Kowalczy- faithfully mimics a function of the proteins in vivo, some kowski 1997a,b). A RecA-mediated strand invasion and mechanism would be necessary to position the RecFR strand exchange then follows, with the resulting crossover complexes near the gaps where they are needed. resolved by some combination of the RuvABC, RecG, Neither RecF protein or RecFR complexes bind and perhaps other enzymes (West 1994). specifically to the ends of DNA gaps in vitro (Webb The chi sites recognized by the RecBCD enzyme et al. 1997; B. Webb & M.M. Cox, unpublished data). A function in only one orientation relative to a RecBCD stalled replication complex would be positioned in part enzyme unwinding and degrading a linear DNA from in the exact location where the RecFR complex would one end (Bianco & Kowalczykowski 1997). In the E. be required to modulate RecA filament assembly, and coli genome, the chi sites are highly over-represented an interaction of RecFR with replication proteins is an (Burland et al. 1993; Blattner et al. 1997; Tracy et al. intriguing possibility. The importance of the RecFOR 1997). Furthermore, most of the chi sites are orientated proteins in a RecA filament assembly can be seen in the so that they would alter the activity of RecBCD RecA-mediated induction of the SOS response, which enzymes moving only in the direction toward oriC is delayed in recFOR mutant cells (Hegde et al. 1995; (Burland et al. 1993; Blattner et al. 1997; Tracy et al. Whitby & Lloyd 1995). 1997). The chi sites are therefore positioned to function Following RecA-mediated DNA strand exchange, in recombinational DNA repair (Kuzminov 1995a). the crossover would be resolved as for double-strand This is true regardless of which template strand is break repair. The RuvABC proteins and the RecG broken. They can modulate the activity of RecBCD helicase provide alternative pathways for the resolution enzymes entering a linear DNA molecule at the site of a of Holliday intermediates (Kuzminov 1996b). The replication-generated double-strand break, and are spaced RuvC protein is a Holliday junction resolvase (West

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modulate RecA filament assembly and disassembly, and the fate of RecA filaments may turn out to be a more complex affair involving interactions with the RuvAB, RecFOR, and perhaps other proteins. Two additional points are worth noting. First, the pathways outlined in Fig. 1 are not necessarily as distinct as shown. For example, some evidence exists that the RecF pathway functions may participate in RecBCD- SSB mediated recombination pathways under at least some RecA conditions (Miesel & Roth 1996). Second, the resolution RecFOR of the recombination crossover in either pathway can occur in two ways. One of the possibilities leads to the formation of a chromosome dimer (Fig. 4). If recombi- national DNA repair is required as often as already postulated, then the formation of dimeric chromosomes should represent a barrier to the segregation of chromosomes at cell division in a large fraction of cells, even under normal growth conditions. This is actually observed, and the problem is addressed by the XerCD site-specific recombination system (step 4 below).

RecOR RecFR RecA Figure 3 Postulated role of RecFOR proteins in RecA filament assembly at a DNA gap. RecA protein does not readily nucleate the filament assembly on single-stranded DNA that is prebound fork undergoing with SSB. A complex of the RecOR proteins facilitates this recombinational a DNA repair nucleation and also prevents the end-dependent disassembly of the filament. Filament extension at the other end is halted by a b b complex of the recFR proteins, bound to duplex DNA near the a gap. resolution path a

1994), and is one of several enzymes with this activity in E. coli (Sharples et al. 1994). A deficiency in RecG protein greatly increases the sensitivity to DNA damage and the recombination defects conferred by ruvABC mutations (Lloyd 1991; Kuzminov 1996b). The RecG protein has helicase activity which promotes the termination of replication migration of a DNA branch or crossover in the direction opposite to that promoted by the RecA protein (Lloyd & Sharples 1993; Whitby et al. 1993; Kuzminov 1996b). The action of RecG following dimeric genome RecA-mediated DNA strand exchange and DNA repair could move the crossover backwards and ultimately reconstruct the framework of a replication fork without the action of RuvC or a similar Holliday resolution to monomers by XerCD system junction resolvase (Kuzminov 1996b). The RuvA and RuvB proteins also displace RecA protein from DNA under some in vitro conditions, suggesting an additional Figure 4 Resolution of a recombination crossover during function for these proteins in vivo (Adams et al. 1994). recombinational DNA repair of a circular chromosome can However, the RecFOR proteins also appear to lead to dimerization of the chromosome.

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Step 3: replication restart following genome (Blattner et al. 1997) has revealed an apparent recombination absence of PAS sites related to those found in the fX174 and ColE1 plasmid origins. Instead, the PriA Just as replication resumes some 30–40 min after the cell protein binds to branched recombination intermediates is exposed to heavy UV irradiation, replication must such as D-loops in order to nucleate the assembly of a resume after recombination under normal growth primosome for replication restart following recombina- conditions. The resumption of DNA replication does tion (McGlynn et al. 1997). The PAS sites found in the not rely on new initiations at oriC, since replication fX174 and ColE1 can take up structures recovers even in a ts dnaA mutant at the restrictive mimicking branched DNA in some respects, and it has temperature (Jonczyk & Ciesla 1979; Khidhir et al. been postulated that they represent an evolutionary 1985). Much genetic evidence links the fX174-type device to expropriate the repair primosome to effect the primosome to this function. As already noted, primo- initiation of viral or plasmid replication (McGlynn et al. some components such as the PriA protein are not 1997). required in oriC-dependent replication. Instead, priA Genetic studies have suggested numerous connec- mutations confer deficiencies in recombination and tions between recombination functions and replica- recombinational DNA repair (Kogoma et al. 1996; tion restart, although the underlying biochemical Sandler et al. 1996). Following recombination, a new mechanisms in most cases remains obscure. The replication fork is assembled in an origin-independent RecG and PriA proteins bind to the same branched manner, mediated in this case by the fX174-type DNA structures, and may compete for binding sites primosome. The genetics suggests a complex interplay in vivo (McGlynn et al. 1997). A deficiency in RecG between recombination and replication functions in protein can be suppressed by mutations that reduce replication restart, although the information available the activity of PriA protein, indicating that the on these interactions is limited. The new replication balance between these proteins is important in vivo forks are distinct from those originating at oriC, at least (Al Deib et al. 1996). The activity of the RecF to the extent that they possess a different primosome for protein, but not the RecOR proteins, is essential in lagging strand DNA synthesis. In effect, the fX174- priA null mutant cells (Sandler 1996), suggesting that type primosome plays a central role in bacterial DNA the RecF and PriA functions overlap. Mutations that metabolism, and might be referred to more descrip- eliminate PriA activity are suppressed by a number of tively, economically and accurately as the repair dnaC mutations, which presumably permit the primosome. loading of DnaB helicase and some kind of primo- Formation of the repair primosome (as defined in some complex in the absence of PriA. Many studies of fX174 replication) requires the PriA, PriB, recombination functions, including the RecA, PriC, DnaC and DnaT proteins, along with DnaB and RecF and RecR proteins, are needed for replication DnaG. The primosome normally assembles in a step- restart (Skarstad & Boye 1988; Smith & Wang 1989; wise manner at the primosome assembly site (PAS) in Livneh et al. 1993; Courcelle et al. 1997; Kogoma fX174 and ColE1 origin DNA (Allen & Kornberg 1997). It is the recombination activities of RecA that 1993; Ng & Marians 1996a,b). PAS is recognized by are required for recombinational DNA repair, as PriA protein, and the PriA-PAS complex is stabilized by opposed to the function of RecA protein in the PriB protein. DnaT protein then joins the complex. induction of SOS (Smith & Wang 1989; Roca & Cox DnaB protein is transferred from a DnaB-DnaC 1990; Asai et al. 1993). In general, it is not clear complex in an ATP-dependent reaction to form a whether the requirement reflects a direct interaction preprimosome containing PriA (2 monomers), PriB between the recombination proteins and the replica- (two dimers), DnaB (one hexamer) and DnaT (one tion apparatus, or the fact that recombination must monomer). PriC (one monomer) is also present, simply precede replication restart. In the case of the although the stage at which it associates is not known. RecF and RecR proteins, it is tempting to speculate The preprimosome can translocate along the DNA in a about the possibility of a direct interaction with the reaction requiring ATP hydrolysis. The transient replication proteins. The recF and recR genes are interaction of DnaG primase with the preprimosome both found in operons that include the genes for produces a completed primosome and leads to the some subunits of DNA polymerase III and other synthesis of RNA primers (Allen & Kornberg 1993; Ng replication functions (Ream et al. 1980; Flower & & Marians 1996a,b). McHenry 1991; Perez-Roger et al. 1991)—perhaps a Analysis of the now-completed sequence of the E. coli coincidence of evolution and perhaps not.

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Step 4: Resolution of dimeric chromosomes bacterial plasmids also contain sites at which the XerCD by the XerCD site-specific recombination enzymes can function and correct the dimerization of system plasmids brought about by homologous genetic recom- bination (Cornet et al. 1994). Excluding the special Recombinational DNA repair involving nearly half of circumstances of conjugation and transduction, little the replication forks originating at oriC should result in occurs within bacterial the formation of numerous dimeric chromosomes via a cells in the absence of DNA damage, even with resolution of the recombination crossover (Fig. 4). The multicopy small genomes (Feng et al. 1991; Hays & conversion of chromosome dimers to monomers is Hays 1991; Cornet et al. 1994; Feng & Hays 1995; mediated by a specialized site-specific recombination Touati et al. 1995). The evolution of plasmid sequences system in E. coli, the XerCD system (Sherratt et al. to take advantage of a host system to resolve dimers 1995). Mutations that inactivate the XerCD site- provides yet another indication of the central place of specific recombinase, or its chromosomal binding site recombinational DNA repair in DNA metabolism. called dif, lead to a lengthening of the average cell, severe filamentation of a substantial fraction of the cells, and abnormal nucleoids (Blakely et al. 1991; Kuempel A unified view of DNA metabolism in et al. 1991). These and other observations have led bacteria several groups to propose that the function of the XerCD system is to convert chromosomal dimers, The replication, recombination and repair of DNA in resulting from recombination, in to monomers (Sher- bacteria have often been presented as distinct topics. A ratt et al. 1995; Kuzminov 1996a). The requirement for useful integration of these processes is evident in a Xer-mediated recombination is reduced greatly in consideration of recombinational DNA repair, as recA¹ cells, reinforcing a connection between the reflected in the related work of many laboratories XerCD system and recombinational DNA repair (Zavitz & Marians 1991; Cox 1993; Livneh et al. 1993; (Blakely et al. 1991; Kuempel et al. 1991). Using a Friedberg et al. 1995; Sherratt et al. 1995; Kuzminov variety of growth conditions, there appears to be a 1996a; Kogoma 1997; Roca & Cox 1997). In many direct relationship between the levels of recombina- respects, the study of bacterial DNA metabolism is tional DNA repair and the requirements for Xer- taking on the integration long evident in the study of mediated site-specific recombination (D. Sherratt, the DNA metabolism of bacteriophage T4 (Karam personal communication). 1994). The outlines of recombinational DNA repair Within the E. coli chromosome, the dif site functions pathways (Fig. 1) offer a way to integrate a variety of only when positioned within a relatively short region phenomena, in addition to those already discussed. near the terminus of replication (Leslie & Sherratt 1995; A system mediating the efficient origin-independent Tecklenburg et al. 1995; Cornet et al. 1996; Kuempel replication restart as a part of frequent recombinational et al. 1996). In this location, it can be replaced by some DNA repair should be manifested in elevated levels of other site-specific recombination systems (Leslie & detectable origin-independent DNA replication under Sherratt 1995). When replication is complete, site- conditions in which DNA is damaged and/or recom- specific recombination at dif sites might proceed bination is stimulated. Phenomena of this kind have repeatedly, with final chromosome segregation occur- been reported, with a particularly detailed characteriza- ring at a point where the chromosomes are monomeric. tion contributed by Kogoma and colleagues (Kogoma Such a model works if the dif sites are positioned within 1997). the last part of the chromosome to be separated at The initiation of replication at oriC is dependent on segregation (Baker 1991). The detailed in vivo function the DnaA protein and requires new RNA and protein of the XerCD system may also rely upon the properties synthesis (von Meyenburg et al. 1979). DNA replication of at least some sequences surrounding dif (Cornet et al. occurring in the absence of protein synthesis is called 1996). stable DNA replication (SDR)(Kogoma & Lark 1970, One of the most striking effects of mutations that 1975). Stable DNA replication is normally repressed, inactivate the XerCD system is the sheer number of but can be induced by conditions that invoke the SOS cells affected. The large fraction of cells with defects in response, giving rise to induced stable DNA replication chromosomal segregation is consistent with the idea (iSDR) (Kogoma et al. 1979). Other forms of stable that a large fraction of replication forks originating at DNA replication observed in some mutant backgrounds oriC undergo recombinational DNA repair. Certain will not be considered here (see Kogoma 1997).

᭧ Blackwell Science Limited Genes to Cells (1998) 3, 65–78 73 MM Cox

Although iSDR is presented as an alternative or back- requirement for defined origins is not absolute. The real up mechanism for the initiation of DNA replication, requirement for iSDR is the introduction of a double- the links between iSDR and recombinational DNA strand break in replicated DNA and the presence of a repair are very evident and acknowledged (Asai et al. nearby chi site (Asai et al. 1994a). Livneh (Livneh et al. 1994a; Kogoma 1997). The requirements for iSDR are 1993) has suggested a unified view in which iSDR and almost identical to those for recombinational DNA replication restart following UV irradiation represent repair and its associated phenomena, such as replication related and overlapping responses to DNA damage. restart following UV irradiation. The chromosomal Under this response, DNA lesions are overcome either origin oriC is not needed. The recombination proteins by the activation of DnaA-independent origins (oriM) RecA, RecBC, RecF and RecN are required for and recombinational repair of stalled replication forks as iSDR, as are the repair primosome components PriA, outlined above. The activation of DnaA-independent DnaB, DnaC, DnaG and DnaT (Kogoma 1997). A origins is simply a matter of providing a double-strand potential requirement for the PriB and PriC proteins break that can be exploited by the vigorous resident has not been explored. The same proteins are intimately recombinational repair system. associated with recombinational DNA repair. One The impact of recombinational DNA repair also distinction that has been noted between iSDR and extends to models for homologous genetic recombina- the repair-associated phenomenon of replication restart tion during conjugation. As already noted, the mechan- is the lack of a requirement for RecBC protein in the isms of conjugational recombination are likely to reflect latter (Kogoma 1997). However, given the multiple the structure of the DNA substrates presented to the cell. pathways for repair prior to replication restart, and the The observation of extensive replication associated with possibility that oriC-mediated replication could con- conjugational recombination (Smith 1991) can be tribute to replication restart, the requirements for viewed as a manifestation of the very active system for recBC could well be a function of the conditions used origin-independent replication restart within recombi- to halt replication and measure its recovery. national repair. The connections can also be seen in the The relationships extend to mechanisms of initiation. requirements for functions such as PriA protein in The mechanism of iSDR initiation is proposed to conjugational recombination (Kogoma et al. 1996). involve the processing of a double-strand break by RecBCD, followed by a RecA-mediated invasion such Finale as that which is envisioned for double-strand break repair in Fig. 1 (Kogoma 1997). The iSDR is The effects of a wide range of mutations in DNA considered a special form of recombination-dependent metabolism functions lead directly or indirectly to the replication. In spite of a reported lack of a requirement same conclusion. Recombinational DNA repair is a for oriC (Kogoma 1997), iSDR cannot be considered a frequent occurrence in cells, even under standard complete back-up replication system. A partially growth conditions. As a central process in DNA replicated chromosome (which must ultimately come metabolism, recombinational DNA repair can be from oriC-mediated initiation) is essential to provide viewed as the primary function guiding the evolution the invading DNA needed to initiate iSDR via of the recombination enzymes, the repair primosome, recombination. De novo replication of a monomeric and the XerCD site-specific recombination system. genome by iSDR is not envisioned. DNA damage is the sometimes unseen cause of The one property of iSDR that is difficult to phenomena affecting every aspect of DNA metabolism. rationalize within the context of recombinational DNA repair is the observed initiation of iSDR at Acknowledgements defined origins (Magee et al. 1992; Asai et al. 1993; Asai et al. 1994b). These origins have been localized very I am grateful to Ken Marians, Andrei Kuzminov and David Sherratt for helpful email discussions during the preparation of close to oriC and the ter (termination) regions of the this manuscript. Described work from the author’s laboratory is chromosome. The stimulation of recombination and supported by grants GM32335 and GM53575 from the National accompanying replication in the ter region might Institutes of Health. simply reflect stalled replication forks as already described. The origins near oriC (called oriM (Kogoma 1997)) are more difficult to rationalize References unless some specialized mechanism exists to introduce Adams, D.E., Tsaneva, I.R. & West, S.C. (1994) Dissociation of double-strand breaks in DNA in the oriM region. The RecA filaments from duplex DNA by the RuvA and RuvB

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DNA repair proteins. Proc. Natl. Acad. Sci. USA 91, 9901– DNA sequence and analysis of 136 kilobases of the Escherichia 9905. coli genome: organizational symmetry around the origin of Al Deib, A., Mahdi, A.A. & Lloyd, R.G. (1996) Modulation of replication. Genomics 16, 551–561. recombination and DNA repair by the RecG and PriA Cao, Y. & Kogoma, T. (1995) The mechanism of recA polA helicases of Escherichia coli K-12. J. Bacteriol. 178, 6782–6789. lethality: suppression by RecA-independent recombination Allen, G.C.J. & Kornberg, A. (1993) Assembly of the primosome repair activated by the lexA (Def) mutation in Escherichia coli. of DNA replication in Escherichia coli. J. Biol. Chem. 268, Genetics 139, 1483–1494. 19204–19209. Capaldo, F.N., Ramsey, G. & Barbour, S.D. (1974) Analysis of the Anderson, D.G. & Kowalczykowski, S.C. (1997a) The recombi- growth of recombination-deficient strains of Escherichia coli K- nation hot spot chi is a regulatory element that switches the 12. J. Bacteriol. 118, 242–249. polarity of DNA degradation by the RecBCD enzyme. Genes Clark, A.J. & Sandler, S.J. (1994) Homologous genetic Dev. 11, 571–581. recombination: the pieces begin to fall into place. Crit. Rev. Anderson, D.G. & Kowalczykowski, S.C. (1997b) The translo- Microbiol. 20, 125–142. cating RecBCD enzyme stimulates recombination by direct- Cornet, F., Louarn, J., Patte, J. & Louarn, J.M. (1996) Restriction ing RecA protein onto ssDNA in a chi-regulated manner. Cell of the activity of the recombination si te dif to a small zone of 90, 77–86. the Escherichia coli chromosome. Genes Dev. 10, 1152–1161. Arai, K. & Kornberg, A. (1981) Unique primed start of phage Cornet, F., Mortier, I., Patte, J. & Louarn, J.M. (1994) Plasmid fX174 replication and mobility of the primosome in a pSC101 harbors a recombination site, psi, which is able to direction opposite chain synthesis. Proc. Natl. Acad. Sci. USA resolve plasmid multimers and to substitute for the analogous 78, 69–73. chromosomal Escherichia coli site dif. J. Bacteriol. 176, 3188– Asai, T., Bates, D.B. & Kogoma, T. (1994a) DNA replication 3195. triggered by double-stranded breaks in E. coli: dependence on Courcelle, J., Carswell-Crumpton, C. & Hanawalt, P.(1997) recF homologous recombination functions. Cell 78, 1051–1061. and recR are required for the resumption of replication at DNA Asai, T., Imai, M. & Kogoma, T. (1994b) DNA damage- replication forks in Escherichia coli. Proc. Natl. Acad. Sci. USA inducible replication of the Escherichia coli chromosome is 94, 3714–3719. initiated at separable sites within the minimal oriC. J. Mol. Biol. Cox, M.M. (1993) Relating biochemistry to biology: How the 235, 1459–1469. recombinational repair function of the recA system is Asai, T., Sommer, S., Bailone, A. & Kogoma, T. (1993) manifested in its molecular properties. BioEssays 15, 617–623. Homologous recombination-dependent initiation of DNA D’Ari, R. & Huisman, O. (1982) DNA replication and indirect replication from DNA damage-inducible origins in Escherichia induction of the SOS response in Escherichia coli. Biochimie 64, coli. EMBO J. 12, 3287–3295. 623–627. Baker, T.A. (1991) . . .and then there were two. Nature 353, 794– Echols, H. & Goodman, M. (1990) Mutation induced by DNA 785. damage: a many protein affair. Mutat. Res. 236, 301–311. Baker, T.A., Sekimizu, K., Funnell, B.E. & Kornberg, A. (1986) Echols, H. & Goodman, M.F. (1991) Fidelity mechanisms in Extensive unwinding of the plasmid template during staged DNA replication. Annu. Rev. Biochem. 60, 477–511. enzymatic initiation of DNA replication from the origin of the Eggleston, A.K., Mitchell, A.H. & West, S.C. (1997) In vitro Escherichia coli chromosome. Cell 45, 53–564. reconstitution of the late steps of genetic recombination in E. Banerjee, S.K., Christensen, R.B., Lawrence, C.W. & LeClerc, coli. Cell 89, 607–617. J.E. (1988) Frequency and spectrum of mutations produced by Feng, W.Y.& Hays, J.B. (1995) DNA structures generated during a single cis-syn thymine-thymine cyclobutane dimer in a recombination initiated by mismatch repair of UV-irradiated single-stranded vector. Proc. Natl. Acad. Sci. USA 85, 8141– nonreplicating phage DNA in Escherichia coli: requirements for 8145. helicase, exonucleases, and RecF and RecBCD functions. Bi, X. & Liu, L.F. (1996) A replicational model for DNA Genetics 140, 1175–1186. recombination between direct repeats. J. Mol. Biol. 256, 849– Feng, W.Y., Lee, E.H. & Hays, J.B. (1991) Recombinagenic 858. processing of UV-light photoproducts in nonreplicating phage Bianco, P.R.& Kowalczykowski, S.C. (1997) The recombination DNA by the Escherichia coli methyl-directed mismatch repair hotspot Chi is recognized by the translocating RecBCD system. Genetics 129, 1007–1020. enzyme as the single strand of DNA containing the sequence Flower, A.M. & McHenry, C.S. (1991) Transcriptional organiza- 50-GCTGGTGG-30. Proc. Natl. Acad. Sci. USA 94, 6706– tion of the Escherichia coli dnaX gene. J. Mol. Biol. 220, 649– 6711. 658. Blakely, G., Colloms, S., May, G., Burke, M. & Sherratt, D. Friedberg, E.C., Walker, G.C. & Siede, W. (1995) DNA Repair (1991) Escherichia coli XerC recombinase is required for and Mutagenesis. Washington, DC: ASM Press. chromosomal segregation at cell division. New Biol. 3, 789– Funnell, B.E., Baker, T.A. & Kornberg, A. (1987) In vitro 798. assembly of a prepriming complex at the origin of the Blattner, F.R., Plunkett, G.R., Bloch, C.A., et al. (1997) The Escherichia coli chromosome. J. Biol. Chem. 262, 10327–10334. complete genome sequence of Escherichia coli K-12. Science Galitski, T. (1996) Evolutionary approaches to the mechanisms of 277, 1453–1474. homologous recombination and mutability. PhD thesis. University Bonner, C.A., Stukenberg, P.T., Rajagopalan, M., et al. (1992) of Utah. Processive DNA synthesis by DNA polymerase II mediated by Galitsky, T. & Roth, J.R. (1997) Pathways for homologous DNA polymerase III accessory proteins. J. Biol. Chem. 267, recombination between direct repeats in Salmonella typhimur- 11431–11438. ium. Genetics 146, 751–767. Burland, V., Plunkett, G.D., Daniels, D.L. & Blattner, F.R.(1993) Hays, J.B. & Hays, J.G. (1991) A probabilistic model for genetic

᭧ Blackwell Science Limited Genes to Cells (1998) 3, 65–78 75 MM Cox

recombination of nonreplicating lambda-phage DNA, stimu- Kuzminov, A. (1995b) Instability of inhibited replication forks in lated by ‘mismatch repair’ of UV photoproducts. Biopolymers E. coli. BioEssays 17, 733–741. 31, 1565–1579. Kuzminov, A. (1996a) Recombinational Repair of DNA Damage. Hegde, S., Sandler, S.J., Clark, A.J. & Madiraju, M.V. (1995) Georgetown, TX: R.G. Landes Co. recO and recR mutations delay induction of the SOS response Kuzminov, A. (1996b) Unraveling the late stages of in Escherichia coli. Mol. Gen. Genet. 246, 254–258. recombinational repair: metabolism of DNA junctions in Horiuchi, T. & Fujimura, Y. (1995) Recombinational rescue of Escherichia coli. BioEssays 18, 757–765. the stalled DNA replication fork: a model based on analysis of Lawrence, C.W., Borden, A., Banerjee, S.K. & LeClerc, J.E. an Escherichia coli strain with a chromosome region difficult to (1990) Mutation frequency and spectrum resulting from a replicate. J. Bacteriol. 177, 783–791. single abasic site in a single-stranded vector. Nucl. Acids Res. Imlay, J.A. & Linn, S. (1986) Bimodal pattern of killing of DNA- 18, 2153–2157. repair-defective or anoxically grown Escherichia coli by Lee, E.H. & Kornberg, A. (1991) Replication deficiencies in hydrogen peroxide. J. Bacteriol. 166, 519–527. priA mutants of Escherichia coli lacking the primosomal Inouye, M. (1971) Pleiotropic effect of the recA gene of replication n0 protein. Proc. Natl. Acad. Sci. USA 88, 3029– Escherichia coli: uncoupling of cell division from deoxyribo- 3032. nucleic acid replication. J. Bacteriol. 106, 539–542. Leslie, N.R. & Sherratt, D.J. (1995) Site-specific recombination Jonczyk, P. & Ciesla, Z. (1979) DNA synthesis in UV-irradiated in the replication terminus region of Escherichia coli: functional Escherichia coli K-12 strains carrying dnaA mutations. Mol. Gen. replacement of dif. EMBO J. 14, 1561–1570. Genet. 171, 53–58. Livneh, Z. (1986) Replication of UV-irradiated single-stranded Karam, J.D. (1994) Molecular Biology of Bacteriophage T4. DNA by DNA polymerase III holoenzyme of Escherichia coli: Washington, DC: American Society for Microbiology. evidence for bypass of pyrimidine photodimers. Proc. Natl. Khidhir, M.A., Casaregola, S. & Holland, I.B. (1985) Acad. Sci. USA 83, 4599–4603. Mechanism of transient inhibition of DNA synthesis Livneh, Z., Cohen, F.O., Skaliter, R. & Elizur, T. (1993) in ultraviolet-irradiated E. coli: inhibition is independent of Replication of damaged DNA and the molecular mechanism recA whilst recovery requires RecA protein itself and an of ultraviolet light mutagenesis. Crit. Rev. Biochem. Molec. Biol. additional, inducible SOS function. Mol. Gen. Genet. 199, 28, 465–513. 133–140. Lloyd, R.G. (1991) Conjugational recombination in resolvase- Kogoma, T. (1997) Stable DNA replication: interplay between deficient ruvC mutants of Escherichia coli K-12 depends on DNA replication, homologous recombination, and transcrip- recG. J. Bacteriol. 173, 5414–5418. tion. Microbiol. Mol. Biol. Rev. 61, 212–238. Lloyd, R.G. & Sharples, G.J. (1993) Processing of recombination Kogoma, T., Cadwell, G.W., Barnard, K.G. & Asai, T. (1996) intermediates by the RecG and RuvAB proteins of Escherichia The DNA replication priming protein, PriA, is required for coli. Nucl. Acids Res. 21, 1719–1725. homologous recombination and double-strand break repair. Louarn, J., Cornet, F., Francois, V., Patte, J. & Louarn, J.M. J. Bacteriol. 178, 1258–1264. (1994) Hyperrecombination in the terminus region of the Kogoma, T. & Lark, K.G. (1970) DNA replication in Escherichia Escherichia coli chromosome: possible relation to nucleoid coli: replication in absence of protein synthesis after replication organization. J. Bacteriol. 176, 7524–7531. inhibition. J. Mol. Biol. 52, 143–164. Madiraju, M.V., Lavery, P.E., Kowalczykowski, S.C. & Clark, Kogoma, T. & Lark, K.G. (1975) Characterization of the A.J. (1992) Enzymatic properties of the RecA803 protein, a replication of Escherichia coli DNA in the absence of protein partial suppressor of recF mutations. Biochemistry 31, 10529– synthesis: stable DNA replication. J. Mol. Biol. 94, 243–256. 10535. Kogoma, T., Torrey, T.A. & Connaughton, M.J. (1979) Magee, T.R., Asai, T., Malka, D. & Kogoma, T. (1992) DNA Induction of UV-resistant DNA replication in Escherichia coli: damage-inducible origins of DNA replication in Escherichia induced stable DNA replication as an SOS function. Mol. Gen. coli. EMBO J. 11, 4219–4225. Genet. 176,1–9. Marians, K.J. (1992) Prokaryotic DNA replication. Annu. Rev. Kornberg, A. & Baker, T.A. (1992) DNA Replication, 2nd edn. Biochem. 61, 673–719. New York: W.H. Freeman & Co. McGlynn, P., Al, D.A., Liu, J., Marians, K.J. & Lloyd, R.G. Kowalczykowski, S.C. (1994) In vitro reconstitution of homo- (1997) The DNA replication protein PriA and the recombi- logous recombination reactions. Experientia 50, 204–215. nation protein RecG bind D-loops. J. Mol. Biol. 270, 212– Kowalczykowski, S.C., Dixon, D.A., Eggleston, A.K., Lauder, 221. S.D. & Rehrauer, W.M. (1994) Biochemistry of homo- von Meyenburg, K., Hansen, F.G., Riise, E., Bergmans, H.E., logous recombination in Escherichia coli. Microbiol. Rev. 58, Meijer, M. & Messer, W.(1979) Origin of replication, oriC, of 401–465. the Escherichia coli K12 chromosome: genetic mapping and Kuempel, P.L., Henson, J.M., Dircks, L., Tecklenburg, M. & minichromosome replication. Cold Spring Harbor Symp. Quant. Lim, D.F.(1991) dif, a recA-independent recombination site in Biol. 43, 121–128. the terminus region of the chromosome of Escherichia coli. New Michel, B., Ehrlich, S.D. & Uzest, M. (1997) DNA double-strand Biologist 3, 799–811. breaks caused by replication arrest. EMBO J. 16, 430–438. Kuempel, P., Hogaard, A., Nielsen, M., Nagappan, O. & Miesel, L. & Roth, J.R. (1996) Evidence that sbcb and recF Tecklenburg, M. (1996) Use of a transposon (Tndif) to obtain pathway functions contribute to RecBCD-dependent trans- suppressing and nonsuppressing insertions of the dif resolvase ductional recombination. J. Bacteriol. 178, 3146–3155. site of Escherichia coli. Genes Dev. 10, 1162–1171. Morimyo, M. (1982) Anaerobic incubation enhances the colony Kuzminov, A. (1995a) Collapse and repair of replication forks in formation of a polA recB strain of Escherichia coli K-12. Escherichia coli. Mol. Microbiol. 16, 373–384. J. Bacteriol. 152, 208–214.

76 Genes to Cells (1998) 3, 65–78 ᭧ Blackwell Science Limited Recombinational DNA repair in bacteria

Ng, J.Y. & Marians, K.J. (1996a) The ordered assembly of the replication in recA mutants of Escherichia coli. J. Bacteriol. 170, phiX174-type primosome. I. Isolation and identification of 2549–2554. intermediate protein-DNA complexes. J. Biol. Chem. 271, Skarstad, K. & Boye, E. (1993) Degradation of individual 15642–15648. chromosomes in recA mutants of Escherichia coli. J. Bacteriol. Ng, J.Y. & Marians, K.J. (1996b) The ordered assembly of the 175, 5505–5509. phiX174-type primosome. II. Preservation of primosome Smith, G.R. (1991) Conjugational recombination in E. coli: composition from assembly through replication. J. Biol. Chem. myths and mechanisms. Cell 64, 19–27. 271, 15649–15655. Smith, K.C. & Wang, T.C. (1989) recA-dependent DNA repair Nurse, P.,Zavitz, K.H. & Marians, K.J. (1991) Inactivation of the processes. BioEssays 10, 12–16. Escherichia coli priA DNA replication protein induces the SOS Sommer, S., Leitao, A., Bernardi, A., Bailone, A. & Devoret, R. response. J. Bacteriol. 173, 6686–6693. (1991) Introduction of a UV-damaged replicon into a Park, E.M., Shigenaga, M.K., Degan, P., et al. (1992) Assay of recipient cell is not a sufficient condition to produce an excised oxidative DNA lesions: isolation of 8-oxoguanine and SOS-inducing signal. Mutat. Res. 254, 107–117. its nucleoside derivatives from biological fluids with a Szostak, J.W., Orr, W.T.L., Rothstein, R.J. & Stahl, F.W. (1983) monoclonal antibody column. Proc. Natl. Acad. Sci. USA 89, The double-strand-break repair model for recombination. Cell 3375–3379. 33, 25–35. Perez-Roger, I., Garcia-Sogo, M., Navarro-Avino, J., Lopez- Tecklenburg, M., Naumer, A., Nagappan, O. & Kuempel, P. Acedo, C., Macian, F. & Armengod, M.E. (1991) Positive and (1995) The dif resolvase locus of the Escherichia coli chromo- negative regulatory elements in the dnaA-dnaN-recF operon some can be replaced by a 33-bp sequence, but function of Escherichia coli. Biochimie 73, 329–334. depends on location. Proc. Natl. Acad. Sci. USA 92, 1352– Ream, L.W., Margossian, L., Clark, A.J., Hansen, F.G. & von 1356. Meyenburg, K. (1980) Genetic and physical mapping of recF Thoms, B. & Wackernagel, W. (1988) Suppression of the UV- in Escherichia coli K-12. Mol. Gen. Genet. 180, 115–121. sensitive phenotype of Escherichia coli recF mutants by recA Roca, A.I. & Cox, M.M. (1990) The RecA protein: structure (Srf) and recA (Tif) mutations requires recJþ. J. Bacteriol. 170, and function. CRC Crit. Rev. Biochem. Mol. Biol. 25, 415–456. 3675–3681. Roca, A.I. & Cox, M.M. (1997) RecA protein: structure, Touati, D., Jacques, M., Tardat, B., Bouchard, L. & Despied, S. function, and role in recombinational DNA repair. Prog. Nucl. (1995) Lethal oxidative damage and mutagenesis are generated Acid Res. Mol. Biol. 56, 129–223. by iron in delta fur mutants of Escherichia coli: protective role of Sandler, S.J. (1996) Overlapping functions for recF and priA in cell superoxide dismutase. J. Bacteriol. 177, 2305–2314. viability and UV-inducible SOS expression are distinguished Tougu, K. & Marians, K.J. (1996) The interaction between by dnaC809 in Escherichia coli K-12. Mol. Microbiol. 19, 871– helicase and primase sets the replication fork clock. J. Biol. 880. Chem. 271, 21398–21405. Sandler, S.J., Samra, H.S. & Clark, A.J. (1996) Differential Tracy, R.B., Chedin, F. & Kowalczykowski, S.C. (1997) The suppression of priA2,kan phenotypes in Escherichia coli K-12 by recombination hot spot chi is embedded within islands of mutations in priA, lexA, and dnaC. Genetics 143, 5–13. preferred DNA pairing sequences in the E. coli genome. Cell Sassanfar, M. & Roberts, J.W. (1990) Nature of the SOS- 90, 205–206. inducing signal in Escherichia coli: the involvement of DNA Umezu, K. & Kolodner, R.D. (1994) Protein interactions in replication. J. Mol. Biol. 212, 79–96. genetic recombination in Escherichia coli. Interactions involving Saveson, C.J. & Lovett, S.T. (1997) Enhanced deletion formation RecO and RecR overcome the inhibition of RecA by single- by aberrant DNA replication in Escherichia coli. Genetics 146, stranded DNA-binding protein. J. Biol. Chem. 269, 30005– 457–470. 30013. Sedgwick, S.G. (1975) Inducible error-prone repair in Escherichia Wang, T.C., Chang, H.Y. & Hung, J.L. (1993) Cosuppression of coli. Proc. Natl. Acad. Sci. USA 72, 2753–2757. recF, recR and recO mutations by mutant recA alleles in Shan, Q., Bork, J.M., Webb, B.L., Inman, R.B. & Cox, M.M. Escherichia coli cells. Mutat. Res. 294, 157–166. (1997) RecA protein filaments: end-dependent dissociation Webb, B.L., Cox, M.M. & Inman, R.B. (1997) Recombinational from ssDNA and stabilization by RecO and RecR proteins. DNA repair—the RecF and RecR proteins limit the J. Mol. Biol. 265, 519–540. extension of RecA filaments beyond single-strand DNA Sharples, G.J., Chan, S.N., Mahdi, A.A., Whitby, M.C. & Lloyd, gaps. Cell 91, 347–356. R.G. (1994) Processing of intermediates in recombination and Wechsler, J.A. & Gross, J.D. (1971) Escherichia coli mutants DNA repair: identification of a new endonuclease that temperature-sensitive for DNA synthesis. Mol. Gen. Genet. specifically cleaves Holliday junctions. EMBO J. 13, 6133–6142. 113, 273–284. Sherratt, D.J., Arciszewska, L.K., Blakely, G., et al. (1995) Site- West, S.C. (1994) The processing of recombination intermedi- specific recombination and circular chromosome segre- ates: mechanistic insights from studies of bacterial proteins. gation. Phil. Trans. R. Soc. Lond. Series B: Biol. Sci. 347, 37–42. Cell 76, 9–15. Shlomai, J. & Kornberg, A. (1980) A prepriming DNA West, S.C., Cassuto, E. & Howard-Flanders, P. (1981) Mechan- replication enzyme of Escherichia coli. II. Actions of protein ism of E. coli RecA protein directed strand exchanges in post- n0: a sequence-specific, DNA-dependent ATPase. J. Biol. replication repair of DNA. Nature 294, 659–662. Chem. 255, 6794–6798. Whitby, M.C. & Lloyd, R.G. (1995) Altered SOS induction Skalka, A. (1974) A replicator’s view of recombination (and associated with mutations in recF, recO and recR. Mol. Gen. repair). In: Mechanisms in Recombination (ed. R.F. Grell), pp. Genet. 246, 174–179. 421–432. New York, NY: Plenum Press. Whitby, M.C., Ryder, L. & Lloyd, R.G. (1993) Reverse branch Skarstad, K. & Boye, E. (1988) Perturbed chromosomal migration of Holliday junctions by RecG protein: a new

᭧ Blackwell Science Limited Genes to Cells (1998) 3, 65–78 77 MM Cox

mechanism for resolution of intermediates in recombination functional role of PriA protein-catalyzed primosome assembly and DNA repair. Cell 75, 341–350. in Escherichia coli DNA replication. Mol. Microbiol. 5, 2869– Wickner, S. & Hurwitz, J. (1975) Association of phiX174 DNA- 2873. dependent ATPase activity with an Escherichia coli protein, Zavitz, K.H. & Marians, K.J. (1992) ATPase-deficient mutants of replication factor Y, required for in vitro synthesis of phiX174 the Escherichia coli DNA replication protein PriA are capable of DNA. Proc. Natl. Acad. Sci. USA 72, 3342–3346. catalyzing the assembly of active primosomes. J. Biol. Chem. Youngs, D.A. & Smith, K.C. (1976) Genetic control. of multiple 267, 6933–6940. pathways of post-replicational repair in uvrB strains of Zyskind, J.W., Svitil, A.L., Stine, W.B., Biery, M.C. & Smith, Escherichia coli K-12. J. Bacteriol. 125, 102–110. D.W. (1992) RecA protein of Escherichia coli and chromosome Zavitz, K.H. & Marians, K.J. (1991) Dissecting the partitioning. Mol. Microbiol. 6, 2525–2537.

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